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Distinct Microbiome in Pouchitis Compared to Healthy Pouches in Ulcerative Colitis and Familial Adenomatous Polyposis Garrett C. Zella, MD, Elizabeth J. Hait, MD, MPH, Tiffany Glavan, BA, Dirk Gevers, PhD, Doyle V. Ward, PhD, Christopher L. Kitts, PhD, and Joshua R. Korzenik, MD Background: Pouchitis occurs in up to 50% of patients with ulcerative colitis (UC) undergoing Heal pouch anal anastomosis (IPAA). Pouchitis rarely occurs in patients with familial adenomatous polyposis (FAP) who undergo IPAA. Our aim was to compare mucosal and luminal flora in patients with UC-associated pouchitis (UCP), healthy UC pouches (HUC), and healthy FAP pouches (FAP). Methods: Nineteen patients were enrolled in this cross-sectional study (nine UCP, three HUC, seven FAP). Patients with active pouchitis were identified using the Pouchitis Disease Activity Index (PDAI). Heal pouch mucosal biopsies and fecal samples were analyzed with a 16S rDNA-based terminal restriction fragment length polymorphism (TRFLP) approach. Pooled fecal DNA from four UCP and four FAP pouches were sequenced for further speciation. Results: TRFLP data revealed statistically significant differences in the mucosal and fecal microbiota between each group of patients. UCP samples exhibited significantly more TRFLP peaks matching Clostridium and Eubacterium genera compared to HUC and FAP pouches and fewer peaks matching Lactobacillus and Streptococcus genera compared to FAP. DNA Sanger sequencing of a subset of luminal samples revealed UCP having more identifiable sequences of Firmicutes (51.2% versus 21.2%) and Verrucomicrobia (20,2% versus 3,2%), and fewer Bacteroidetes (17,9% versus 60.5%) and Proteobacteria (9,8% versus 14,7%) compared to FAP.
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Conclusions: The pouch microbial environment appears to be distinctly different in the settings of UC pouchitis, healthy UC, and FAP. These findings suggest that a dysbiosis may exist in pouchitis which may be central to understanding the disease. Abdominal colectomy with ileal pouch anal anastomosis .(IPAA) is the preferred surgery for patients with ulcerative colitis (UC) unresponsive to medical therapy or with dysplasia and for those with familial adenomatous polyposis syndrome (FAP),I Inflammation of the ileal pouch, termed pouchitis, is the most common long-term complication in patients with UC who undergo IPAA.2 Pouchitis occurs in up to 50% of these patients3'4 and is the major factor leading to morbidity after IPAA surgery in patients with UC.3't In comparison to individuals with a history of UC, those undergoing IPAA for FAP are far less likely to develop pouchitis.5 The pathogenesis of pouchitis is incompletely understood. Proposed etiologies include fecal stasis, bacterial overgrowth, dysbiosis, genetic susceptibility, short-chain fatty acid deficiency, ischemic complications of surgery, bile-acid toxicity, immune alteration, a missed diagnosis of Crohn's disease (CD), or a recurrence of UC in the pouch.1.6,1 Pouch bacteria appear central to the pathophysiology of the disease. The efficacy of antibiotics in ameliorating pouchitis underscores the role of mucosal or luminal bacteria in the development of pouchitis." The role of gut microbes in pouchitis is further supported by studies revealing fewer pouchitis relapses and postoperative prophylactic benefit with probiotic therapy.1° A therapeutic utility of both antibiotics and probiotics suggests there may be subtle shifts in pouch microflora that culminate in pouch inflammation. Recently, Komanduri et all' described a pouch dysbiosis in five patients with UC and pouchitis when compared to patients with UC and no pouchitis and to healthy ileal tissue in continuity with the colon. Inflamed pouch mucosa was found to have greater bacterial species diversity than pouch
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control or healthy ileal tissue. Fusobacterium varium was more abundant in pouchitis tissue, while Streptococcus species were dramatically reduced. Healthy UC pouches possessed a unique floral pattern, with more representatives from the Clostridium, Enteric, and Streptococcus groups compared to ileal tissue from healthy subjects. In our study we aimed to characterize both mucosal and luminal microflora in inflamed UC pouches compared to healthy UC and FAP pouches. We designed the study with FAP pouch control subjects in an effort to find shifts in flora that were distinct to UC and inflammation rather than fecal stasis. We utilized 16S ribosomal DNA gene-based terminal restriction fragment length polymorphism (TRFLP) techniques to characterize luminal and mucosal microbial environments, followed by DNA sequencing for further characterization and validation. MATERIALS AND METHODS Patient Population Individuals over age 18 years were enrolled from the Massachusetts General Hospital (MGH) Gastrointestinal Unit in one of three cohorts with pouches after IPAA: those with a history of UC and subsequent pouchitis (UC pouchitis, UCP), those with a history of UC without pouchitis (healthy UC pouch, HUC), or those with a history of FAP without pouchitis (healthy FAP pouch, FAP). The diagnosis of UC and FAP in these patients was based on standard clinical, radiologic, and histologic diagnostic criteria. UCP patients were eligible if they had experienced at least two episodes of antibiotic-responsive pouchitis symptoms in the previous 2 years or had persistent pouchitis symptoms despite two courses of antimicrobial therapy. A Pouch Disease Activity Index (PDAI, comprising clinical, endoscopic, and histologic elements) score >7 was required for inclusion in the UCP group.I2 Eligible UCP patients underwent colectomy with IPAA more than 3 months prior to enrollment. Eligible HUC and FAP patients underwent colectomy with IPAA more
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than 1 year prior to enrollment without experiencing pouchitis symptoms. Exclusion criteria for each group included any indication of CD and the use of probiotics, biologic or other immunosuppressive therapy, rectal therapy, or new antibiotics during the previous 2 months. Chronic antibiotic use for more than 3 months was permitted in the UCP group if the patient's symptoms persisted without the use of new antibiotics. Data Collection Key clinical data collected from each patient included age, gender, disease duration and extent, pouch duration, number of pouchitis episodes in the previous year, medication use, and the clinical components of the PDAI. The PDAI clinical score takes into account stooling frequency and the presence of hematochezia, stooling urgency, abdominal cramping, and fever. Endoscopic and histologic findings were noted for each patient and incorporated into the PDAI score. Specimen Collection All patients underwent pouchoscopy after clinical data were collected. Patients received a short course of bowel preparation 1 day prior to the procedure with phospho-soda. Luminal pouch contents were suctioned through the colonoscope into a sterile container. Approximately I cc of the suctioned stool was aliquoted into each of three vials containing 1 cc of 10% glycero1/90% sterile water. Tubes were inverted to generate a homogeneous mixture then immediately placed in dry ice. The pouch was examined for elements of the PDAI endoscopic score: edema, granularity, friability, loss of vascular pattern, mucous exudates, and ulceration. Then three biopsies were taken at the most inflamed sites or at random locations from normal-appearing tissue. Each tissue specimen was immediately rinsed in normal saline then placed in a vial containing 1 cc of 10% glycerol/90% sterile water. The vials were inverted to submerge the tissue
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specimen then immediately placed in dry ice. All specimens were then stored at —80°C within 30 minutes. TRFLP Analysis All stool and tissue specimens were analyzed by TRFLP at the Environmental Biotechnology Institute at California Polytechnic State University (CPSU). DNA was isolated using MoBio's Power Soil DNA extraction kit according to the manufacturer's protocol (MoBio Laboratories, Carlsbad, CA). Triplicate 0.1-g samples were used for the extraction of stool samples, while the entire biopsy sample was used for the isolation of DNA from tissue samples. Bacterial 16S ribosomal RNA genes were amplified from both types of sample using the forward primer 8DF (5'-AGA GTT TGT TCM TGG CTC AG-3') and the reverse primer 536-K2R (5'-GTA TTA CCG CGG CTG CTG G-3'). The forward primer was fluorescently labeled with a phosphamide dye. Fifty microliter reactions were assembled using 1 FL of undiluted DNA, 5 pl., of 10x buffer, 3 pl. of 10 mM dNTPs, 2 tL of 20 mg/mL BSA, 7 pL of 25 mM MgCl2, 1 tL of each primer, and 0.3 ,td, of 5 U/L TaqGold (Applied Biosystems, Foster City, CA). Reaction temperatures and times were as follows: 95°C for 10 minutes followed by 30 cycles of 95°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes, followed by a final extension of 72°C for 10 minutes. The quality of the extractions and polymerase chain reaction (PCR) reactions were confirmed using gel electrophoresis. PCR triplicates were combined during a PCR cleanup performed using MoBio's PCR Cleanup Kit following the manufacturer's protocol (MoBio Laboratories). PCR products were quantified using a FLX800 microplate fluorescence reader tuned to the labeling dye (Bio-Tek Instruments, Winooski, VT).
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Three enzyme digests were independently performed using cleaned-up PCR product and the restriction endonucleases HaeIH, Hpall, and Alul (New England Biolabs, Beverly, MA). Each 40-1,tL digest included 75 ng DNA, 1 U enzyme, and 4 pi, buffer. Samples were digested for 4 hours at 37°C and the enzyme was inactivated for 20 minutes at either 65°C (HpaII and Alul) or 80°C (Haell1). Resulting fragments were ethanol precipitated and resuspended in 20 [IL formamide and 0.25 tL CEQ 600 basepair standard. Terminal restriction fragments were separated using capillary gel electrophoresis and profiles were obtained using a Beckman Coulter (Fullerton, CA) CEQ8000X DNA analysis system. Terminal restriction fragment lengths and relative peak areas were exported from the CEQ8000 into Excel (Microsoft, Seattle, WA). TRF data consists of peak sizes, which indicate the length of the DNA fragment in nucleotides, and the area under each peak, which provide a mea-sure of abundance for each DNA fragment. Since the amount of DNA loaded on the capillary cannot be accurately controlled, the sum of the total peak area varies between TRF patterns. Therefore, peak areas are normalized by converting the value to parts per million to standardize the data for comparison." Peaks with an area less than 10,000 ppm (<1.0% of the total) were excluded from the analysis to reduce excess noise. Data from three independent restriction enzyme digests were included to reduce the incidence of distinct sequences with equivalent TRF lengths. TRF fragments determined to differ most between sample groups were compared to available GenBank (Bethesda, MD) sequences to tentatively identify potential bacterial populations. TRFLP data were analyzed for peaks that could be identified by database analysis as representing particular groups of organisms. 16S Ribosomal DNA-based DNA Sequencing Four fecal samples from each of the UCP group and the FAP group were selected for sequencing analysis of pooled DNA. Samples with the greatest intersample similarities in TRF
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profiles (as determined with Bray—Curtis similarity indices) were selected in an effort to create the most uniform pooled specimens representative of each patient group. DNA from the samples was processed as detailed for TRF analysis, using full-length 16S PCR primers without fluorescent label, then sent to the Broad Institute (Cambridge, MA) for sequencing. Samples were cloned into the pCR2.1-TOPO vector and sequenced on an ABI3730 DNA sequencer. Sequences were trimmed using LUCY.14 Read pairs from each clone were then assembled using the following alignment-assisted assembly method implemented at the Broad Institute. A reference sequence for use as an alignment template for the read pairs was first selected from a core set of nonchimeric 16S rRNA sequences obtained from Greengenes.15 The core set reference sequence sharing the greatest number of matching k-mers with the aggregate k-mer set of both forward and reverse reads was selected. Forward and reverse reads were each aligned to the core reference sequence using Blast.I6 Aligned forward and reverse reads were then assembled based on the alignment with base quality scores used and preserved through assembly. For purposes of classification, only assembled sequences of greater than 1100 nucleotides were considered. Sequences were classified using a naïve Bayesian rRNA classifier." The classifier was trained on the Ribosomal Database Project core set, RDP10.I8 Statistics Differences in patient characteristics were assessed using Fisher's Exact test for categorical variables and the Exact Kruskal—Wallis test for continuous variables. TRFLP datasets were transformed by taking the square root of the area under each peak to deemphasize large TRFLP peaks while still accounting for relative abundance. Transformed data from UCP, HUC, and FAP groups were compared using Bray—Curtis similarity, multidimensional scaling, and analysis of similarity (ANOSIM) (Primer E, Plymouth, UK). ANOSIM was also used to compare
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intersubject and intrasubject TRFLP profile similarities. Analysis of variance (ANOVA) was used to compare relative abundance of specific TRFLP pattern elements between each group of subjects. Among the pooled fecal DNA samples, differences in the percentage of total identifiable clones represented by each bacterial genus were calculated using Fisher's Exact test, considering that the number of identifiable clones in a sample is proportional to species abundance. Ethical Considerations This study was approved by the Institutional Review Boards at MGH and CPSU. [Insert Table 1] RESULTS Patients A total of 19 patients were enrolled and divided into three groups: UCP (n = 9), FAP (n =. 7), and HUC (n =- 3) (Table 1). Overall, 37% of patients were female and the average age was 40.6 years. There were no significant differences in baseline demographics or disease characteristics between the three groups. PDAI values were significantly higher in the UCP group (P < 0.001). All mucosal biopsies in the UCP group displayed histologic evidence of active pouchitis. One UCP patient received chronic ciprofloxacin therapy for the prior 8 years and one received chronic rifaximin therapy for the prior year, both with continued episodes of pouchitis. Three UCP patients had received short courses of ciprofloxacin ranging from 3 months to 1 year prior to enrollment. The remaining four UCP patients received ciprofloxacin briefly —2 years prior to enrollment.
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TRFLP Analysis Significantly more peaks were observed in TRFLP data from mucosal samples (average total number of peaks/ sample in three digests = 64.8, standard deviation [SD] = 12.2) compared to fecal samples (average total number of peaks/sample in three digests = 41.3, SD = 8.2) with P < 0.001 (Fig. 1). The greatest amount of similarity existed within samples from the same person, with both mucosal and fecal sample similarity greater within a subject than between subjects (P = 0.027). There was no significant difference between patient groups in the degree of similarity between fecal and mucosal samples (P > 0.05). Between individuals, mucosal TRFLP profiles were found to be more consistent than fecal samples (P < 0.001). TRFLP data from mucosal and fecal samples were significantly different between each of the three patient groups. Figure 2 displays the results of multidimensional scaling analysis comparing TRFLP data. Significant differences (P < 0.05) in bacterial TRFLP data were revealed between the UCP group, HUC group, and FAP group when comparing mucosal samples, fecal samples, and when both sample types were combined. Samples from the two UCP patients receiving a single long-term antibiotic were not significantly different compared to other UCP samples. Samples from the three patients who received prior short courses of antibiotics within 1 year were also not significantly different, as all nine UCP samples grouped together apart from HUC and FAP. Approximately 15% of the total number of TRFLP peaks were matched to peak sets in the database. In an analysis of TRFLP elements that represent bacterial genera, a set of TRFs matching Lactobacillus and Streptococcus (Hae 264-5, Hpa 97-9, Alu 76, 532) were present at higher relative abundance in both mucosal and fecal samples from FAP patients compared to UCP (ratio 5:1 in mucosa, 3:1 in stool). A second set of TRFLP peaks (Hae 272-4, Hpa 222-3, Alu 440) matching Clostridium, Eubacteriurn and Roseburia genera were present at five times the
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relative abundance in stool from UCP patients compared to FAP patients. Fecal samples from HUC pouches also had fewer peaks matching Clostridium, Eubacterium, and Roseburia compared to UCP (ratio 1:15). HUC stool revealed fewer Escherichia, Streptococcus, and various sulfur-oxidizing bacteria (Hpa 496, Hae 205, Alu 74) at a ratio of 1:2 compared to UCP. Mucosal samples from HUC pouches revealed a similar smaller quantity of Escherichia, Streptococcus, and various sulfur-oxidizing bacteria compared to UCP (ratio 1:2). [Insert Figure 1 and Figure 2] DNA Sequencing In all, 2304 clones were processed for each pooled sample (4608 total clones). After data processing and analysis, good quality forward and reverse reads were identified with a reliable classification at the genus level (>80%). A total of 712 sequences were identified in the UCP pooled sample and 1015 in the FAP pooled sample. At the phylum level, the UCP pooled sample revealed significantly more Firmicutes and Verrucomicrobia (52% and 22% of clones, respectively) compared to the FAP group comprised of 19% Firmicutes and 3% Verrucomicrobia (P < 0.001 and P < 0.001, respectively). However, numbers of clones in the Bacteroidetes phylum were significantly higher in the FAP group (71% of clones) compared to UCP (20% of clones, P < 0.001). There were no significant differences between pooled samples among the Fusobacteria, Proteobacteria, and Actinobacteria phyla (Table 2). [Insert Table 2] Multiple bacterial genera within the class Clostridia were significantly more prominent in the UCP group compared to the FAP group (Table 3). Among Clostridia, the UCP group had more clones represented by the genera Roseburia (8% of clones in UCP versus near 0% in FAP, P < 0.001), Lachnospiraceae Incertae Sedis (28% versus 10%, P < 0.001), Clostridium (9% versus 1%, P < 0.001), Lachnospira (2% versus 0, P < 0.001), and Veillonella (3% versus near 0%,
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P < 0.001). The UCP pooled sample also had more clones from the genus Prevotella than the FAP group (6% versus 1%, P < 0.001). Finally, the UCP group revealed 22% of its pooled clones from the genus Akkermansia in the phylum Verrucomicrobia, compared to only 3% in FAP (P < 0.001). Members of the genus Bacteroides predominated in the pooled fecal sample of the FAP group, with 678 of 1015 (67%) clones representing Bacteroides, compared to 99 of 712 (14%) in UCP (P < 0.001). Other genera that were statistically more prominent in the FAP group were Faecalibacterium in the family Ruminococcaceae (6% of clones in FAP, 0% in UCP, P < 0.001), Parabacteroides (2% in FAP, 0% in UCP, P < 0.001), and Escherichia (2% in FAP, 0% in UCP, P < 0.001). DISCUSSION The results of our study suggest that the pouch microbial environment is distinctly different between patients with UC-associated pouchitis and healthy UC or FAP pouches. Using 16S ribosomal gene-based TRFLP data, we identified significant differences in bacterial communities between all three patient cohorts in both stool and mucosa. These broad differences in TRFLP profiles were further explored using DNA sequencing. Sequencing revealed multiple statistically significant variations in specific bacterial genera between pooled fecal DNA from a subgroup of patients with UCP and a subgroup of patients with FAP. We report data that supports previously published work suggesting a dysbiosis is central to the development of pouchitis. The efficacy of both antibiotics and probiotics in ameliorating the symptoms of pouchitis underscores the theory that shifts in bacteria may be important in the gener-ation and cessation of pouch inflammation."" However, few details are known about the precise bacterial populations in the pouch and the nature of the dysbiosis associated with pouchitis.
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Studies based on classic culturing techniques have suggested the presence of more anaerobes in pouch effluent compared to ileostomy effluent and that pouch fecal bacterial populations are similar to rectal flora.2022 A later study using similar stool culture techniques noted a greater number of facultative anaerobes in the inflamed pouch lumen compared to the noninflamed UC pouch or normal ileum.23 More recently, Duffy et a124 used culture-based techniques to show that sulfate-reducing bacteria were present in the stool of 8 of 10 healthy UC pouches but not in any of seven FAP pouches. Molecular techniques have been used to define the pouch microbiota more precisely. Falk et a125 used TRFLP to demonstrate that pouch mucosal species in two healthy UC pouches resembled those from the colon. However, populations of Clostridium perfringens in both patients were unique to the pouch. To our knowledge, only one study, by Komanduri et al,E I used molecular techniques to compare pouch microbiota between UC patients with and without pouchitis. Using length-heterogeneity PCR followed by sequencing, they concluded that inflamed pouch mucosa had greater species diversity and more Fusobacterium varium than healthy UC pouches or healthy deal tissue in continuity with the colon. Noninflamed UC pouches revealed more representatives from the Clostridium, enteric, and Streptococcus groups compared to pouchitis and healthy nonpouch Heal tissue. These differences between UC and health could be explained by the fact that the healthy ileal tissue was in continuity with the colon without fecal stasis. In each of these studies the healthy pouch control subjects suffered from UC, without any non-inflammatory bowel disease (IBD) pouch controls. Our study was designed to utilize molecular techniques to identify bacterial populations unique to inflamed or healthy UC pouches compared to a non-IBD pouch, keeping pouch fecal stasis as a consistent factor across all patient groups. This design and our findings differed from the results of Komanduri et al.1' We found no increase in Fusobacterium species among inflamed
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pouches and instead noted the largest numbers of Clostridium species in the UCP group. We also noted a unique reduction in Bacteroides in the inflamed pouch compared to FAP. Our findings suggest that a mucosal and luminal dysbiosis exists in pouchitis, not only when compared to the healthy UC pouch but also when compared to a non-IBD pouch. Additionally, healthy UC pouches differed significantly from FAP pouches. This suggests an alteration in ileal pouch microbiota that may be unique to the UC disease state, with or without inflammation. Our TRFLP results supported an overall increase in fecal Clostridium in UCP patients compared to FAP patients. This finding may be consistent with those of Falk et al.25 DNA sequencing confirmed our findings, with the genus Clostridium representing 9% of identifiable clones in the UCP pooled sample compared to only 1% in the pooled FAP sample. More broadly, the class Clostridia accounted for 53% of identifiable clones in the UCP group compared to 21% in the FAP group. The genus Eubacterium was also more prevalent in the UCP stool by TRFLP, but no known Eubacterium species were revealed by sequencing. This may be due to the difficulty of TRFLP to distinguish between Eubacterium and Clostridium species. The decrease in Lactobacillus and Streptococcus genera in UCP compared to FAP pouches also was not reflected in the sequencing data. However, this is not surprising since the number of clones identified as representing Lactobacillus and Streptococcus was less than 0.5% of the total identified. These disparities between TRFLP and sequencing results could be explained by our sampling only four patients from each group for sequencing. Importantly, TRFLP is best used to compare bacterial community structure between samples and is less precise than sequencing analysis for bacterial speciation.26 Sequencing results revealed an overall decrease of the phylum Bacteroidetes in the inflamed pouch. This finding is consistent with multiple molecular surveys of intestinal microbiota in both CD and UC.27-3° Bacteroidetes play a key role in maintaining gut health, so it has been
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proposed that a relative reduction in this population may favor the development of inflammation.29 Consistent with our TRFLP data, DNA sequencing also demonstrated a significant increase in clostridia in the inflamed pouch, namely among the genera Clostridium, Lachnospiraceae, and Rosehuria. While multiple studies have shown a relative decrease in clostridia species among patients with CD,30-32 our findings are consistent with previous work in UC revealing more clostridia in inflamed UC rectal mucosa compared to noninflamed mucosa.33 While evidence to date supports a general shift in multiple species as being linked to the development of IBD, we note two particular genera that reflect specific theories of IBD pathogenesis. We found significantly greater numbers of the genus Roseburia among inflamed pouches compared to FAP pouches. Roseburia are flagellated commensal inhabitants of the colon. Flagellin have been shown to induce proinflammatory gene expression by activating Toll-like receptor 5 (TLR5), and patients with TLR5 polymorphisms and low levels of antiflagellin antibodies may be protected from developing CD.34-36 High levels of antiflagellin antibodies, specifically anti-CBirl antibodies to flagellin of Clostridium species, are present in 50% of CD patients, 6% of UC patients, and may be associated with the development of pouchitis.37'38 It is unknown whether Roseburia species may play a role in TLR5 activation leading to increased mucosal inflammation. Additionally, we found the mucin-degrading genus Akkermansia of the phylum Verrucomicrobia to be significantly more prevalent in pouchitis. Several theories exist regarding the role of mucin in the protection of intestinal epithelium in IBD, with mutations, alterations, and degradation of mucins being associated with CD and UC.39-42 A recent large-scale genome-wide association study revealed that a gene encoding for mucin proteins, MUC19, was mutated significantly more often in patients with CD.43 While the mucin-degrading Verrucomicrobia species and Akkermansia in particular have been identified within the human
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colon,44'45 there have been only two reports of DNA from these bacteria in patients with CD" and no previous reports of Verrucomicrobia in UC. This study has certain limitations. TRFLP is best used to compare bacterial community structure between cohorts and is not precise in identifying species. Thus, the results of TRFLP should be interpreted as suggestive of bacterial groups, rather than specific species, and emphasis should be placed on the separation between each group. The study may have found more differences between patient groups if the sample size had been larger. Nevertheless, we were able to identify statistically significant differences in TRFLP data and clone libraries between groups. Another limitation was the pooling of DNA into groups for DNA sequencing. Individual sequencing of each sample was limited due to expense. This may have led to an apparent difference in flora between groups if only one sample had a unique bacterial profile. However, we grouped samples with similar TRFLP data in an effort to minimize the chance of one sample skewing the analysis. Additionally, the use of antibiotics in UCP patients may have altered gut flora. However, TRFLP profiles were similar in all UCP patients and distinct compared to HUC and FAP, regardless of whether the subjects had received antibiotics chronically or briefly 1-2 years before enrollment. This suggests that the dysbiosis may be unique to pouchitis and not a direct result of exposure to antibiotic medications. In conclusion, we identified differences in the pouch microbiome between the inflamed pouch in UC, the healthy pouch in UC, and the healthy pouch in FAP. These differences in bacterial populations were evident in stool and mucosa by TRFLP analysis. 16S rDNA sequencing revealed specific differences in luminal bacterial genera, with the pouchitis group having substantially fewer Bacteroidetes and more Clostridia compared to the healthy FAP group. These findings reinforce other surveys of the micro-biome in both UC and CD. Additionally, one genus with flagellated species and one genus with mucin-degrading species were more prevalent in the
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Stunning Tuscany
... waving his winner's flag all evening. - Watching the most immense storm while overlooking the main square , just about keeping dry while eating fabulous Bruschetta.Incredible experience. I am now writing this in the cool of the evening with the fresh smell of rain in the air. I am overlooking the Tuscan hills and walls of Siena with a Limoncello waiting for me.... Oh yes ... and the highlight????? ..,,, The kids are in ...
Montisi and Hot air ballooning
One of the highlights of the trip so far was our morning ballooning with Chris and Claire from "Ballooning in Tuscany". We had an early start (5am) to be down at the launch field for 5.40am and then we took off at about 6.30-6.45am on a beautifully sunny morning with fantastic views over the Tuscan countryside - the pictures hopefully do it more justice than any description I can ...
Twilight's Montepulciano and Volterra
... no fountain.
As I said on FB, I joked with Lare that I would give him $100 if he would pose in the same archway as Edward, unbuttoning his shirt as Edward did. I had to pay up--isn't he fabulous? Total. Keeper. (Even though he doesn't sparkle,)
Hidden underground passageway like the one the Volturi used to skulk down to their palatial lair.
The archway Bella ran through during the parade to get to Edward in time ...
Last Day in Montepulciano
... wine with biscotti and you dunk the biscuits in the wine, eat the bickies as they go soft, and then drink the wine with the crumbs and all. YYYUUUMMMM. Definitely something I am going to add to my dinner menu.
I dined with four gorgeous, young, incredibly intelligent and fun people who left me for dead with their knowledge and wide range of interests. One minute they are laughing at cat recordings they had seen on u tube to discussing ...
Determination pays as discover 5 star piscina!!
My final day in Cortona and I have a last walk around the township, stopping in the piazza to see what's going on, sitting at Tuscher cafe in the main street trying one of Massimos famous Shakerato's (shaken cold coffee)...then popping into the old boxed seat theatre of which I would've loved to spend an evening here being entertained...it's tiny, dramatically curved and absolutely gorgeous. The ceiling is painted a baby blue (although it's come out green in my ...

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I almost walked past this beautiful leaf. I was focused on something different, probably some flowers or birds. My husband noticed it though and he pointed it out to me.
As it turned out, it's one of my favorite photographs that I've taken. It's not actually a leaf but a ghost of one.


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The Magazine
The Lessons of Alito
It's important to remember one thing: quality matters.
Feb 6, 2006, Vol. 11, No. 20 • By TERRY EASTLAND, FOR THE EDITORS
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The Democrats have yet to find a red state where they can make the case for what might be called "living-Constitution" jurists. But there can be no doubt that the debate over judicial selection will intensify both this year and again in 2008. What's unknown, of course, is whether Bush's last years in office will offer him another opportunity to pick a justice. If so, it's not too early to say that he should select another in the mold of Roberts and Alito.
-Terry Eastland, for the Editors
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text: A Simpler Way to Estimate the Feedback Between Permafrost Carbon and Climate One of the big unknowns in predicting climate change is the billions of tons of carbon frozen in Arctic permafrost. As global warming causes soil temperatures to increase, some of this carbon will decompose and enter the atmosphere and accelerate climate change.... How the brain builds new thoughts Let's start with a simple sentence: Last week Joe Biden beat Vladimir Putin in a game of Scrabble. It's a strange notion to entertain, certainly, but one humans can easily make sense of, researchers say, thanks to the way the brain constructs new thoughts. A new... The psychology behind religious belief: Religion fulfills 16 basic human desires Throughout history, scholars and researchers have tried to identify the one key reason that people are attracted to religion. Some have said people seek religion to cope with a fear of death, others call it the basis for morality, and various other theories abound.... Sex causes immune system changes that increase chances of conception Research from Indiana University has found that sexual activity triggers physiological changes in the body that increase a woman's chances of getting pregnant, even outside the window of ovulation. The results could eventually influence recommendations regarding how... Many Atolls May be Uninhabitable Within Decades Due to Climate Change A new study shows that the combined effect of storm-induced wave-driven flooding and sea level rise on island atolls may be more severe and happen sooner than previous estimates of inundation predicted by passive "bathtub" modeling for low-lying atoll islands, and... Researchers use digital pill to study medication adherence Researchers at UMass Medical School will soon begin a pilot study to explore how a digital pill would allow doctors to monitor patients who do not take their medicine as prescribed in real-time–a major health care problem in the United States. "As many as 20 percent... From Our BloggersA Spinal Emulator for Medical Training [The Brain Bank Manc]Brightness-equalized quantum dots improve biological imaging [Engineering at Illinois]Summary of the Sixth BHD and First International Upstate Kidney Cancer Symposium [BHD Research]The Sea Life is Never the Same [The Poetry of Science]EPA institutes new oil-refinery-emissions rules, tougher standards set [Air Quality Matters]Growth & Aging; Creatine & Health [Josh Mitteldorf]What navigating the VW-diesel-vehicle-recall road ahead may look like [Air Quality Matters]Thinking on your feet: The effects of dance on the brain [The Brain Bank Manc]Promise of Novel Alzheimer's Treatments [Josh Mitteldorf]Live Update: Sixth BHD & First Upstate Kidney Cancer Symposium [BHD Research]Drought does or does not make air pollution worse; which is correct and why? [Air Quality Matters] Many Atolls May be Uninhabitable Within Decades Due to Climate Change A new study shows that the combined effect of storm-induced wave-driven flooding and sea level rise on island atolls may be more severe and happen sooner than previous estimates of inundation predicted by passive "bathtub" modeling for low-lying atoll islands, and... Researchers use digital pill to study medication adherence Researchers at UMass Medical School will soon begin a pilot study to explore how a digital pill would allow doctors to monitor patients who do not take their medicine as prescribed in real-time–a major health care problem in the United States. "As many as 20 percent... Flame Retardant Breakthrough is Naturally Derived and Nontoxic Inspired by a naturally occurring material found in marine mussels, researchers at The University of Texas at Austin have created a new flame retardant to replace commercial additives that are often toxic and can accumulate over time in the environment and living... Tanja Bosak Microbe sleuth Microbes may seem small and insignificant, but in evolutionary terms the tiny organisms punch far above their weight. Although only a few micrometers in size, microbes have been responsible for shaping much of the world around us, including the air that we breathe and... Inferotemporal cortex: How the brain recognizes objects When the eyes are open, visual information flows from the retina through the optic nerve and into the brain, which assembles this raw information into objects and scenes. Scientists have previously hypothesized that objects are distinguished in the inferior temporal... Scientists grow organic semiconductor crystals vertically for first time Our smartphones, tablets, computers and biosensors all have improved because of the rapidly increasing efficiency of semiconductors. Since the turn of the 21st century, organic, or carbon-based, semiconductors have emerged as a major area of interest for scientists... Arsenic found in many U.S. red wines, but health risks depend on total diet A new University of Washington study that tested 65 wines from America's top four wine-producing states — California, Washington, New York and Oregon — found all but one have arsenic levels that exceed what's allowed in drinking water. The U.S. Environmental... Females more at genetic risk for developing insomnia than males Genes may contribute more to the development of insomnia symptoms in females than in males, according to a new study led by a Virginia Commonwealth University graduate student. Drawing on pre-existing data from the Virginia Adult Twin Studies of Psychiatric and... Dormant viral genes may awaken to cause ALS Scientists at the National Institutes of Health discovered that reactivation of ancient viral genes embedded in the human genome may cause the destruction of neurons in some forms of amyotrophic lateral sclerosis (ALS). The results, published in Science Translational... Scientists use holography to peer into clouds Watching clouds go by, swirls of white puff up and melt away. The changes mirror mixing within the clouds as drier air mingles with water-saturated air. New research led by scientists at Michigan Technological University, along with the National Center for Atmospheric... Priests found spiritual satisfaction by serving nuns The culture of medieval chivalry conjures tales of valiant knights vowing to honor and protect fair damsels. Yet outside the realm of romance, men regarded women as their inferiors – or so we thought. According to Stanford historian Fiona Griffiths, "We've... Novel Tag Developed for Squid, Jellyfish Invertebrates, such as squid and jellyfish, play a crucial role in the marine food web and are also vital commercial fisheries. Despite their importance, little is known about their natural behaviors or how their environment influences those behaviors or physiology.... Projects explore Internet-enabled manufacturing The National Science Foundation (NSF) has provided more than $6.5 million in awards for research exploring ways to expand and ease access to manufacturing services. In 30 projects involving 28 universities, faculty and students will research, prototype and test new... High-fructose diet hampers recovery from traumatic brain injury A diet high in processed fructose sabotages rats' brains' ability to heal after head trauma, UCLA neuroscientists report. Revealing a link between nutrition and brain health, the finding offers implications for the 5.3 million Americans living with a traumatic brain... Researchers probe the physical forces involved in creating the mitotic spindle Many millions of times per day, football-shaped structures called mitotic spindles form within the body's cells as they prepare to divide. The process is routine but mysterious, as the micro-mechanics involved are not yet well understood. In research published October... More Science News Make It So


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BP PLC said cost of the oil spill response amounted to about $450 million as of May 13, and scientists and engineers from across industry and government continued work to stop an oil spill from a blowout in 5,000 ft of water off Louisiana.
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A day of two halves
... trip and worth every penny.
We returned to our hotel and then spent the afternoon walking or using the tram and monorail to explore the hotels along the strip. They are stunning and `larger than life` sort of places. The Venetian was incredible with a complete replica of St. Marks Square and canals with gondolas. It is difficult to describe them so hopefully the photos will help.
From here we returned to our hotel to ...
Not Yet...
... to be completed by about 8 AM or it's WAY too hot to finish.
Two weekends ago, we headed up to Great Basin National Park for the new moon astronomy program. The stars were incredible! I got some great shots of the Milky Way rising over the mountains. It was also a good test of the new CBs that Chris and I had installed in our trucks. The reviews are positive, and we're excited with how well we can stay in touch using the CBs instead ...
Viva Las Vegas - Days 63 - 66
... and shadows of the red rocks were beautiful in the afternoon sun. We noticed how it was so difficult to guess our speed and altitude once we were away from the city, you also couldn't feel the acceleration of the helicopter at all. Danny pointed out all the landmarks including the stunning Hoover dam and he was also great at telling us when any turbulence was on its way, which Kate appreciated! It was a very different sensation to flying, and thankfully one that we all ...
"When I met you in the summer"
... smile. I can die happy now lol. The funny thing was Jess didn't even realise he was British and just thought I was waving my passport around like an idiot! The passport was stowed away safely and we went back to dancing by the pool.
As always with every perfect day, it was stopped early with a bit of torrential rain. We ran into the casino and started to make our way out. As we were going up the escalator a bouncer was throwing our shirts ...
Vegas part 1
... bed to lie on. I am really looking forward to getting to San Francisco so I can get rid of some of my camping clothing and sleeping bag to lighten the load a bit. I also can't wait to do laundry! It's the little things I guess. I am still struggling to comprehend that this is just the start of my 3 month trip. It's quite overwhelming to think about anything beyond this week. I'm looking forward to having a week of time on my own I think. I find ...


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CLEMSON, S.C. -- Willy Korn came to Clemson in 2007 as a can't-miss prospect. He'll leave this spring simply wanting to prove he can still play football.
The Tigers backup quarterback said Thursday he'll transfer to Marshall after graduating in May and hopes to compete for a starting job at the Conference USA school.
The Thundering Herd went 7-6 last season, beating Ohio 21-17 in the Little Caesar's Pizza Bowl. Korn said that Marshall's new coach, John "Doc" Holliday, didn't guarantee a starting spot, just an opportunity.
"I feel good about the situation up there and as long as I continue to work hard the rest of the semester and go up there, be myself and play to the best of my ability, I feel good about winning the job," Korn said.
NCAA rules permit Korn to go from one FBS school to another as long as he's gotten his degree, and the graduate major he'll take at his new school is not offered at his old one. Korn will major in journalism, a graduate-level program Clemson does not offer.
He'll finish 20 hours of course work in communications, work out in Clemson's weight room and keep sharp with receivers this spring. Korn will also work at least once a week with Bobby Bentley, his former Byrnes High School coach.
Korn was one of South Carolina's biggest recruiting prizes in 2007 after leading the Rebels to two state championships. Korn had his heart set on the Tigers early on -- he committed at the start of his junior season.
With a friendly demeanor and a winning smile, Korn quickly became a fan favorite even before he officially enrolled in January 2007.
When he made his on-field debut at Clemson that fall, Korn got a standing ovation from the Death Valley crowd, upstaging a record-setting, five-touchdown effort from starter Cullen Harper.
Injuries kept Korn on the sidelines most of that season, and he expected to play sparingly again in 2008 behind Harper. He emerged when Tommy Bowden called on him to start against Georgia Tech with the Tigers struggling at 3-3. Bowden walked away from the job a few days later and interim coach Dabo Swinney stuck with Korn as the starter.
Korn was hit on the arm, an injury that required offseason surgery that sapped his strength and disrupted his mechanics.
He struggled to keep up with rising freshman Kyle Parker, who eventually won the job and had a record-setting season in leading Clemson to its first ACC Atlantic Division title in 2009.
Korn says there are plenty of times he wonders what might've been had he not gotten injured against the Yellow Jackets. "I don't think about it so much any more because I'm focused on what's ahead," he said. "But I'd be lying if I said I didn't think about that, especially then."
Korn says Marshall was the only Bowl Subdivision school he looked at. The Thundering Herd opens at Ohio State on Sept. 2
Korn says he's got no regrets about choosing Clemson or bitterness about how things played out.
"I did absolutely everything I could. I worked as hard as I could've done and things just didn't work out," he said. "It's not like I'm the only guy it happened to. I'm just fortunate and lucky I get a second chance to go and win a job somewhere."


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The head of the government's civil liberties protection board said Thursday that its classified review of the NSA's collection of Americans telephone records didn't turn up any evidence of abuses — but both he and the man who lead the National Security Agency's program said it's still time to end bulk collection.
Retired Air Force Gen. Michael V. Hayden, who was NSA director when the spy agency began the bulk phone-records collection program under the Bush administration, said he supports the bill winding through Congress that would end the government's ability to collect and store phone metadata, instead leaving the information with the phone companies, which would only turn over limited data approved by court order.
Both Mr. Hayden and David Medine, the chairman of the Privacy and Civil Liberties Oversight Board, also said they support creating an independent advocate for average Americans' interests before the secret court that oversees intelligence programs.
SEE ALSO: Liberty vs. security in post-9/11 world
The two men spoke alongside Sen. Mike Lee and two top officials from the American Civil Liberties Union at a panel on privacy sponsored by The Washington Times, the ACLU and Microsoft. Times Opinion Editor David A. Keene moderated the discussion, which saw Mr. Hayden say the NSA can live within the new constraints — but the public will see a drop in security.
"Draw the box [and] we'll play inside the smaller box," Mr. Hayden said. "By increasing your comfort level, you've also almost certainly increased your danger level. But as long as you're comfortable with that, that's the social contract. That's the way democracies work."
The NSA program came to light after former government contractor Edward Snowden leaked its details last year. The government has since acknowledged it is collecting and keeping five years' worth of telephone metadata — the times, durations and parties involved in phone calls — and searching it to try to spot terrorist links.
SEE ALSO: Protect yourself from being a hacker's easy target
Since then, questions have been raised about the extent and efficacy of that information, gathered on Americans under Section 215 of the Patriot Act, as well as information gathered on foreigners under Section 702 of the Foreign Intelligence Surveillance Act.
The House last month passed a bill that would halt bulk collection and leave the data in the hands of phone companies. The NSA could still compel the companies to turn over big chunks of data but would have to justify each request by citing specific "selector terms."
Still, that bill was watered down from the original version at the behest of the Obama administration and with the compliance of House GOP leaders.
"I think the USA Freedom Act is an important first step," said Laura W. Murphy, director of the ACLU's Washington Legislative Office, who said she hopes the Senate adds back in some of the protections dropped in the House version. "It's attacking just the tip of the iceberg when it comes to reining in mass surveillance."
Mr. Hayden said that when the phone program began, it collected nearly 100 percent of metadata records. But lately it's been about 25 or 30 percent.
He said the new bill will change that.
"It's going to be a little cumbersome. It's going to be a little harder to hop from Verizon to AT&T to some other phone company in order to do the chaining," he said. "But in return for the lack of agility, we will now have access to all of your phone records, and I think that is a happy compromise."
Mr. Hayden repeatedly pushed back against what he said was a misunderstanding of the programs and the law.
He said one misconception is to say bulk data collection is an executive program. He said Section 215 of the Patriot Act was passed by Congress and reauthorized even after bulk collection was going on and said it's done with the approval of the secret Foreign Intelligence Surveillance Court.
"In the 215 program you've got all three branches going check, check, check. That's kind of the Madisonian trifecta. That's how it's supposed to work," he said.
He said there have been no abuses of the bulk data collection program — and was challenged by the ACLU's Ms. Murphy, who interjected, "That we know of."
Mr. Hayden then pointed to Mr. Medine, who, as chairman of the oversight board, had classified access. "He's looked at the program [and] he says there's no abuse."
Mr. Medine didn't dispute that, but he said there's reason to be "cautious" the more data the government is given and how it's used. He cited the Census Bureau's collection of information that the government used during World War II to round up Japanese Americans for internment.
Mr. Medine also questioned the need for the metadata bulk collection in the first place. His board's report contradicted the intelligence community's initial assertions that it had prevented some potential terrorist attacks through the bulk collection program.
"The phone metadata program [isn't needed] because there are alternative ways of getting access to the program that serve the same, and maybe even better, ends," he said.
Mr. Hayden said that's true — for now.
"It's better because we can afford more time. It may not have been the right answer in 2002. But now [it may be right] given what we know of al Qaeda, the degree of threat, all the other intelligence tools we have — and that's an important point," Mr. Hayden said.
For his part, Mr. Lee questioned the current status of court decision-making on bulk data, saying he doubted whether prior Supreme Court precedent anticipated, or is appropriate for, a 21st century level of technological exchange — and the government's ability to monitor it.
"What might have been stored on papers and personal effects inside our physical houses in the 1790s would not necessarily be put onto a piece of tangible paper today," said Mr. Lee, a Utah Republican. "Nonetheless, these same privacy concerns still need to be protected, and I think some of the programs we're talking about today threaten to undermine those same privacy and security interests that are at the heart of the Fourth Amendment."
He said the question is more complex than just a straight question of constitutionality because no final court has yet ruled the programs unconstitutional — and given various rules, it's "difficult to conceive of the precise procedural route" that a case would take to reach the Supreme Court.


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Town Square
Pleasanton Weekly wins awards in California newspaper contest
Original post made on May 7, 2012
Read the full story here Web Link posted Monday, May 7, 2012, 7:42 AM
Comments (2)
a resident of Lund Ranch II
on May 7, 2012 at 9:17 am
Congratulations on winning the newspaper awards...
Having a website of our own, I am always interested in what makes a winning website - www.PaloAltoOnline.com .The video of the Palo Alto parade, at the top of the site, caught my eye immediately.
Is there any chance the Pleasanton Weekly will start using videos in their online version too?
a resident of Highland Oaks
on May 7, 2012 at 3:29 pm
Congratulations and keep up the good work!
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Editorial: Pleasanton Police Department's unwarranted silence unacceptable
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League is preparing for change as the RFL decides which 14 clubs will compete at the top level for the next three years, writes Andy Wilson
As he makes his Super League exit, Andy Wilson says giant Tongan winger, Lesley Vainikolo, will leave a remaarkable trail behind him.
John Kear runs the rule over "the next Stuart Fielden": 18-year-old prop Sam Burgess.
According to Andy Wilson Terry Newton's recent disciplinary hearing highlights the need for a more transparent procedure.
About 41 results for Sportblog + Bradford Bulls
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Danny Edwards BBQAugust 21, 2014BBQ • Kansas City, United States
The Big D is awesome! Thick and tender; a tough combination to do.
The Big D is awesome! Thick and tender; a tough combination to do.
Spicy pork soft tacos and shredded beef soft tacos are awesome!
Tacos al pastor are the best in Kansas City.
Get the brisket sandwich. You won't regret it.
Best tailgating there is.
Most things here are brand-name items in identical packaging with a different name. Great deals, but be careful on some prices that cost more than their original brands.
There are only three voting booths, so be prepared.
They really know their stuff at this one.
Get the Prime Rib sandwich. It's awesome!
Awesome, knowledgeable staff and new equipment. Not an overcrowded social club. The members make it a very welcoming environment and nobody is looked down upon.


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Alere Photos
Alere ReviewsAlere 195
"Ok place to work "
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Great people to work with.
Long hours with not much opportunity for advancement
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I applied online. The process took 2 weeks. I interviewed at Alere (Orlando, FL) in August 2015.
Normal interview process, talk with recruiter first, then with hiring manager. Very easy to talk to, made me feel at ease. Asked appropriate questions. The company's communication throughout the entire process was great.
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When you really need an answer -- fast -- toss out the crystal ball and bring in Alere (formerly Inverness Medical Innovations). Alere offers both professional and consumer diagnostic tests as well as health management services. Its professional diagnostic products include tests for cancers, cardiovascular disease, drugs of abuse, infectious diseases, and women's health including pregnancy tests and fertility monitors. The firm's Alere Health division provides health management services including wellness programs, disease monitoring, and care coordination for ... More


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This Just In
Lexus made a drivable cardboard car that's 100 percent recyclable.
Innovative vehicles and the technology under their hoods represent a staggering leap forward in the evolution of cars.
Wow! The SUV accelerates from zero to 60 miles in 3.2 seconds!
For years, Volkswagen strived to produce and sell more motor vehicles than any other manufacturer in the world, putting quantity over quality.
VW equipped cars with software that turns off emissions controls when driving and turns them on during an emissions test.
Auto-braking technology can reduce insurance injury claims by as much as 35 percent.
The Frankfurt Motor Show opens in Germany on September 15, and several concept cars caught our eye. Have a look for yourself.
Here's a hint -- they're critical for human drivers to see during a rainfall.
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Goals from Alvaro Morata, Isco and Alvaro Vasquez secured what was an easy win in the end against a much changed Dutch team.
Germany beat Russia in the last group game, an irrelevant match in terms of qualification with both teams already out.
What is your opinion? Feel free to discuss the article under. You have to comment by using your Facebook profile. Be fair and have respect for other people and opinions!
Who of Europe's top strikers will move to England this summer?
After a dreadful start to their Bundesliga campaign, Borussia Dortmund appear to have weathered the storm after going five matches without defeat.


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I've never mailed hundreds of thousands of dollars worth of marijuana to anyone, but if I were going to do it, I'd at least make sure that it was properly addressed. But that's apparently not the case for whomever sent 90 pounds of pot to one Philly-area clothing store over the course of two days. [More]
Someone Sent $270K Worth Of Pot To The Wrong Clothing Store
Bob Marley's Family Is Starting A Line Of Branded Marijuana For Reasons No One Must Explain
In a branding combination that goes together like Bob Marley black light posters and college students, the late musician's family says it's starting a new line of Bob Marley marijuana. If you need someone to explain the relationship between Marley and Mary Jane, go ask your 19-year-old niece/nephew/son/daughter living in the basement. [More]
Of Course There's An App For Getting Medical Marijuana Delivered To Your Doorstep In L.A.
As marijuana becomes legal in a growing list of states, whether recreationally or for medical reasons, it would make sense that consumers living in those areas would turn to technology to get the products they want. After all, who actually calls the delivery place on the phone to get dinner anymore? Calling a cab, how quaint! So to fill that technology need, a California company has set its app up to offer medical marijuana delivery. [More]
Voters In Oregon, Alaska, Washington D.C. Legalize Marijuana
Break out the celebratory brownies: Alaska, Oregon and D.C. are joining the ranks Washington State and Colorado, after residents had their say in last night's vote and legalized marijuana where they live, in varying degrees. [More]
Dressing Your Baby In A Marijuana Leaf Costume: Totally Chill Or Completely Inappropriate?
On the one hand, marijuana is legal for medical and recreational use in some states in our fair country, and the plant's leaf is just another green thing that grows in the ground. But on the other hand, babies and marijuana. [More]
Colorado Health Officials Pushing To Ban Most Forms Of Edible Marijuana
With Halloween coming up, law enforcement officals Colorado have been warning parents about pot-laced candies or treats that might find their way into kids' bags this year, now that recreational marijuana is legal in the state. And in order to decrease the likelihood of pot product confusion in the future, health officials are now pushing the state to ban most edible forms of pot, including brownies, cookies, cakes and most candies. [More]
Hermès Customers Returning $20,000 Birkin Bags For Smelling Like Weed
When you're paying $20,000 for a purse, it should be basically perfect, if not possess some kind of supernatural ability to lay golden eggs or something. That's why some disgruntled Hermès customers are reportedly returning their pricy Birkin bags, claiming that the purses smell like stanky, skunky marijuana. [More]
Washington Stores Pull Exploding Marijuana Soda Bottles From Shelves
Now that marijuana is legal for recreational use in Washington, stores have probably not been surprised to see pot-infused products flying off the shelves. But when there's soda literally exploding on the shelves, well, that's not the best way to clear out the inventory. At least three marijuana product purveyors in the state have had to pull a sparkling pomegranate soda made with pot after bottles started exploding mysteriously. [More]
Seattle Prosecutor Dropping Seven Months Worth Of Tickets For Using Marijuana In Public
Remember that cop in Seattle accused of going rogue in light of Washington's legalization of recreational marijuana? Officials in that city say he issued about 80% of all the tickets for using pot in public, and had urged ticketed residents to contest any and all marijuana tickets as a result. Now Seattle's prosecutor is just wiping seven months of marijuana tickets from the board entirely. [More]
Washington State Just Says No To Mobile Weed-Smoking Wagons
In the wake of Washington state's recent legalization of recreational marijuana, some businesses have popped up to cater to weed tourists who want to see the sites while also enjoying the cannabis they have purchased. But state regulators have now made it clear that smoking pot on buses — even chartered ones — is a no-no. [More]
Seattle City Attorney: Anyone Who's Gotten A Ticket For Marijuana Should Fight It
After the Seattle Police Department announced at the end of July that a police officer had been reassigned and was under investigation for issuing 88% of the city's marijuana citations, the city attorney is telling anyone who got a ticket to contest it. [More]
Cannabis Company Unveils Country's First Marijuana Billboards In Seattle
Back in my day, the only billboards along the side of the road were for things like light beer, ambulance chasers, adult video stores, fast food and car dealerships. No longer will roadside advertising be limited to such things, as a new era of everyday legal marijuana use in Washington has made the country's first cannabis billboards a reality. [More]
When Your Parents Don't Let You Make Pot Brownies At Home, Don't Break Into A Stranger's House
First of all, let us acknowledge that it is unclear what kind of drugs two teens allegedly baked into brownies at an absent stranger's house. But based on the pop culture knowledge we possess here at Consumerist (Have you seen Half Baked? That Dave Chappelle is going places!), we're gonna go ahead and guess that they were making pot brownies. Allegedly. [More]
Colorado Marijuana Regulators Propose Emergency Rule To Clarify Potency Of Pot Edibles
When a New York Times columnist is writing that she hallucinated that she was dead after eating more than the recommended dose of edible marijuana, while other consumers perhaps unused to judging the potency of pot are also complaining about confusing serving sizes, there's a bit of pressure on Colorado regulators to come up with a solution. That's why officials are reportedly prepping an emergency rule that would make it easier to tell how much pot is in edible pot products. [More]
Seattle Police Officer Reassigned After Review Finds He Wrote 80% Of City's Marijuana Tickets
While marijuana has been legal in Washington state since the beginning of this year, it's not like the streets are filled with people lighting up bongs and toking on pipes. Because if you do, you're going to get a ticket for public marijuana use. That being said, the Seattle Police Department says one officer has been reassigned after apparently going a bit ticket-happy and issuing about 80% of the city's pot citations so far this year. [More]
Oregonians Get Another Chance To Vote On Recreational Marijuana Come This November
For the last two years, Oregonians in favor of marijuana for recreational use have had to stare glumly across the border at Washington State, where it's legal, after a ballot initiative to legalize the stuff failed in 2012. But if at first you don't succeed… You know where this is going. [More]
Washington State's Legal Marijuana Shops Prepare To Open Their Doors This Week
Like Colorado before it, Washington State is now open for legal marijuana sales. With the first licenses for the first stores issued today — and a required 24-hour waiting period before sales can start — shop owners are preparing to welcome customers this week. [More]
Customer Surprised Because Sonic's Menu Doesn't List Chicken Strips With A Side Of Marijuana
Today seems to be the day when fast food customers end up with surprises in their food, like Cracker Jack! But not as fun/innocent: First it was a fried hand towel at KFC, and now a Sonic customer says she found a plastic baggie of marijuana in her order of chicken strips and fries. [More]


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Hourly - Expert ($$$) - Est. Time: 3 to 6 months, Less than 10 hrs/week - Posted
Info pad project . 1. 8.5 by 11.5 inch screen, non-touch 2. 4G or LTE communications capable 3. One USB port 4. Wi-Fi enabled 5. 1 Gig of Ram 6. Excellent Video performance for multimedia video files (Video Card) 7. 2 Speakers 8. 1 Audio Out Port – Ear Phones (sound control) 9. Base on Android OS or linux distribution (ubuntu) or if you have a better idea. 10. 64 gig (flash hard drive) 11. See if you can use www.sunstone.com pcb123 desigh software Working page change arrows on the side of the device. Mutidirectional arrow key on the botom of the device.(to cange the content from news paper to magazine or mutimedia content program) And the accept key or enter button on the butom of the device. Component Selection in Design Teams hands.(price quality) Waithing for a quote and i hope we can manage a longterm agrement if everything works out.


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Children's Theatre CompanyNovember 18, 2013Theater • Minneapolis, United States
Fabulous presentation of Cinderella! Enjoyable for all ages!
Fabulous presentation of Cinderella! Enjoyable for all ages!
Gluten free Caesar salad $17. Literally 2 cups of romaine & 4 oz of steak. No cheese, no croutons, nothing else. $17. Ridiculous!
So many great gluten free options!!!
Staff is super friendly and seem to be knowledgable about snakes.
Not sure why it's labeled as a smoke shop... Pretty poor selection of craft beers. Have yet to find a liquor store in Chaska with a decent variety.
I'm confused as to why, after I told the server we were wondering if we could substitute items because we can't have gluten, she proceeded to ask each of us if we wanted pancakes, muffins, or toast.


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"After Earth" - Trailer (M. Night Shyamalan, Will & Jayden Smith)
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June 6 2013, 02:48 PM
Agent Richard07
Re: "After Earth" - Trailer (M. Night Shyamalan, Will & Jayden Smith)
Joerib wrote:
Seen it, not a bad movie, not a blockbuster for sure!! I think most of the hate for this film is the Director!! (maybe a lil Will Smith also lol)
Yeah, I saw it the other day and it wasn't a bad movie. I figured too that maybe people have a bias against Shyamalan. I didn't see anything that made it a terrible movie. In fact, I even liked the message about keeping the mind clear so that fear doesn't take hold. It's a central theme in a lot of religions and philosophies and has little to do with Scientology. My only gripe is that I watched expectling a twist or revelation at the end and didn't get one, but that's on me, not the movie itself.
Agent Richard07
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Sharing your career news/moves in the "On the Move" sections of local business journals, industry trade publications and even your alumni magazines is a great way to keep the business community abreast and engage your professional network. However, too often news of this sort is sent out without a photo. Editors have a twin objective: deliver the news, and do so in a layout that is visually appealing. When given the choice between two similar news items, one with a photo and one without, the one with will win every time.
Let's delve into some of the reasons why photos aren't being sent or aren't being used when they are sent:
1) Photos are out-of-date. We have all seen the black-and-white '80s mugshots that some professionals so desperately hold onto, perhaps in light of an ever-dwindling hairline (something I know well) or intractable insistence that they not be photographed again. We have actually seen situations in which the "master" takes are physical, not digital. Your clients and potential clients deserve to see the you of today, not the you of the Reagan Era.
2) High-res versions are nowhere to be found. The photos that appear on your website are vastly scaled-down versions of a high-resolution shot. The full-size photo (sometimes slightly scaled-down) is what publications need. If you search-and-search and can only find the tiny pictures, you need to take a new photo. A good photographer should provide you with full-scale and small-scale photos. The real issue is that you can always size a large photo down without losing quality, but you can never do the reverse.
3) Senior leaders refuse to be photographed. A high-quality headshot needs to be a prerequisite for all public-facing, senior-level professionals. By not setting – and holding individuals – to this standard, you are sending a strange message to clients. Frankly, you want to avoid the, "I think he is a recluse," comments. Photographs personalize relationships, ever-important in an increasingly remote world, and convey subtle messages such as confidence and trustworthiness.
4) The only photos we have are from the old firm or personal collection. The former is a copyright issue and should be avoided. The latter is rife with issues ranging from poor resolution to cropping out other people in a photo.
So, what should you do if you have news ready-to-go and no photo to accompany it? Wait. Non-pressing news will simply be more impactful if paired with a pictorial component. One strategy to avoid these sorts of scenarios is to create an "On the Move"/new hire checklist and add an entry for securing a high-res photo. A picture may not be worth a thousand words, but it will help your item merit greater attention.
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DISCLAIMER: Because of the generality of this update, the information provided herein may not be applicable in all situations and should not be acted upon without specific legal advice based on particular situations.
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Source: Bengals 'far apart' on contract with draft pick Rivers
Updated: August 4, 2008, 12:36 PM ETBy James Walker | ESPN.com
The Cincinnati Bengals and first-round pick Keith Rivers are still "far apart" in contract negotiations, a source told ESPN.com Sunday. Rivers, the No. 9 overall pick in April's NFL draft, has missed the entire first week of training camp with Cincinnati.
The two sides remain in constant negotiations. The source added that talks could improve dramatically at any time, but as of Sunday afternoon the team was not close to striking a deal with Rivers.Jacksonville Jaguars rookie ...
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|Publication number
|Publication type
|Application number
|Publication date
|14 Aug 2003
|Filing date
|8 Feb 2002
|Priority date
|14 Mar 2001
|Also published as
|Publication number
|Jeffrey Moritz, Allan Avnet
|Original Assignee
|Beckman Coulter, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (5), Referenced by (18), Classifications (7), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
[0001] The present invention relates generally to an electrically conductive plastic rack for pipette tips, and more particularly, it relates to an electrically conductive plastic rack for use with pipette tips following removal of the pipette tips from a plurality of pipettes.
[0002] Modem laboratory automation often involves conducting experiments using plates having multiple wells. These plates are known as "microplates". A typical microplate may contain three hundred and eighty four (384) wells or more. The microplate wells are used to hold specimens to be tested in a laboratory experiment. Pipettes are used to transfer liquids in and out of the microplate wells during the laboratory experiments. For example, the pipettes may transfer the initial specimens into the wells along with a number of different chemical reagents used during the laboratory experiment.
[0003] To prevent contamination of the pipettes when transferring liquid into and out of the microplate wells, a disposable polypropylene pipette tip is attached to the end of each pipette. The pipette tip carries all of the liquid to be transferred from one location to another, and prevents the actual pipette from contacting the liquid. After the transfer, the pipette tip is removed from the pipette and a new pipette tip is attached to the end of the pipette for subsequent transfers of different liquids. In this manner, precise concentrations, ingredient mixtures and amounts of liquids may be transferred using each pipette without the fear of contamination from a previous liquid transfer using the same pipette.
[0004] Multiple pipette devices are often used when conducting experiments using multiple well microplates. For example, a robotic device having three hundred eighty four (384) pipettes may be used when conducting experiments using a three hundred eighty four (384) well microplate. Such multiple pipette devices provide for the rapid removal or insertion of liquid into every microplate well. When using multiple pipette devices, tip racks are used to hold the pipette tips to be attached to the ends of the pipettes. The tip racks hold the tips in an organized fashion and allow the tips to be quickly inserted onto the pipettes or removed from the pipettes. Like the pipette tips, the tip racks are typically formed of polypropylene for ease of manufacture and cost savings. Because the tips and racks are both formed of relatively low cost polypropylene, it is customary in the industry to discard the tips and racks after a short period of time, or even after one use, for disposal and/or recycling.
[0005] With reference now to FIG. 1, the head 20 of a multiple pipette device 24 is shown. The head 20 holds three hundred eighty four pipettes 22 used to transfer liquid to and from a three hundred eighty four well microplate 50. Pipette tips 30 are shown inserted on each pipette 22 of the multiple pipette device 24. The pipette tips are positioned above a tip rack 40 used to hold the pipette tips 30 when they are removed from the pipettes 22 of the multiple pipette device 24.
[0006] Before the pipettes may be used to transfer liquid to and from the microplate wells, the tips 20 must be loaded onto the pipettes. A robotic arm (not shown) typically delivers a rack 40 full of tips 30 to the loading area 42. The head then positions itself directly above the rack 40 with each pipette in alignment with one of the tips 30. The head 20 then moves downward to bring the pipettes into contact with the tips 20. Each tip 20 is secured to a pipette with a friction-fit. Thus, the head must press (or be pulled) down with a substantial force to cause the tips to slide on to the ends of the pipettes. The sliding action of the polypropylene tips against the metal mandrels causes an electrostatic charge, i.e., triboelectricity, to develop on the exterior of the tips 20.
[0007] Once the tips are loaded onto the pipettes 22, the head 20 moves the pipettes and connected tips 30 to position the tips in the wells of the microplate 50. The pipettes then suck liquid from the wells and into the tips 30 for delivery to another location. After transferring liquid from one location to another (e.g., one microplate to another), the head returns to a position above the rack 40 so the head 20 is in position to return the used tips 30 to the rack. A shuck plate (not shown) then forces the tips from the pipettes, causing the tips 30 to fall into the tip rack 40 such that each tip is positioned in a tip seat on the tip rack. Of course, as the tips 30 are forced from the pipettes, they slide along the pipettes, once again causing an electrostatic charge to be generated and deposited upon the tips as a result of the rubbing action between the tips and the pipette. After the tips 30 are returned to the tip rack 40, the robotic arm may remove the rack full of tips for disposal or cleaning. Alternatively, the tips and rack may remain in place on the loading area for further use by the multiple pipette device.
[0008] Unfortunately, once the tips are returned to the rack, the triboelectric effect often creates a problem with the tips in the tip rack. In particular, when the polypropylene tips are returned to the polypropylene rack they each contain an electrostatic charge as a result of the rubbing action between the pipettes and tips during loading and unloading of the tips on the pipettes. The polypropylene tip rack does not conduct electricity. Thus, the static charges remain on the tips as they sit in the rack because there is no flow of the electric charge from the tips to the tip rack. The static charges on different tips and the rack itself cause some of the tips to repel away from each other and the rack. When the electrostatic charge is sufficient, this repelling action may cause one or more of the light-weight tips to become dislodged from the proper tip seats within the rack. In the worst situations, one or more tips may jump completely out of the tip rack as a result of the electrostatic forces between tips. When a tip becomes dislodged from a tip seat or jumps completely out of the tip rack, significant problems arise because loose tips may be lost and not properly discarded and removed from the testing area. Also, if in a subsequent step of a laboratory process the head is scheduled to return to the tip rack and once again join the pipettes to the used set of tips, an improperly seated tip on the tip rack may be crushed by the force of the head as the head moves the pipettes into position to pick up the tips. This may result in operational failure of the multiple pipette device and/or faulty experiment results.
[0009] For the foregoing reasons there is a need for a multiple pipette system that will prevent tips from becoming displaced in the tip rack as a result of the triboelectric effect during loading and unloading of the tips. In addition, there is a need for a tip rack for use with a multiple pipette system that will prevent electrically charged tips from becoming displaced within the rack.
[0010] The present invention is directed to an apparatus that satisfies the need for a tip rack for use with a multiple pipette system that prevents electrically charged pipette tips from becoming displaced within the tip rack. The apparatus is a tip rack comprising a face having a plurality of seats formed thereon for holding pipette tips and at least one sidewall depending from the face. The face and the at least one sidewall of the tip rack are integrally formed as a one-piece conductive outer shell. The tip rack further comprises a support insert connected to the conductive outer shell such that the support insert is covered by the conductive outer shell. The support insert includes a plurality of support walls that form chambers dimensioned to receive the pipette tips.
[0011] The face and the at least one sidewall are comprised of an electrically conductive plastic material whereby static electricity deposited on the pipette tips is discharged through the tip rack when the pipette tips contact the tip rack. A number of different electrically conductive plastic materials may be used to form the tip rack. For example, the electrically conductive plastic material may include polypropylene impregnated with carbon, metal flakes, metal powder or metal strands. In one preferred embodiment of the invention, the tip rack comprises about 15% or more carbon, by weight, and preferably, about 21% carbon, by weight.
[0012] When pipette tips having an electric charge deposited thereon are contacted with the tip rack, the electric charge deposited on the pipette tips is conducted through the electrically conductive tip rack and either delivered to ground or dissipated on the tip rack. Thus, when the pipette tips are seated in the tip rack, the pipette tips will not be displaced in the tip rack because any significant electrostatic charges on the pipette tips are conducted away from the pipette tips and through the tip rack. These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and the appended claims.
[0013]FIG. 1 shows a perspective view of a multiple pipette device holding tips positioned over a tip rack;
[0014]FIG. 2 shows a perspective view and cutaway portion of the conductive plastic rack for pipette tips.
[0015] With reference to FIG. 2, a conductive plastic tip rack 60 for pipette tips is shown. The conductive plastic rack includes a conductive outer shell 64 formed around a support insert 62. The support insert 62 is manufactured separate from the conductive outer shell 64, and the two pieces are designed to easily fit together to form the complete rack 60. For example, the support insert 62 and conductive outer shell 64 may snap-fit, or be joined using other connection means (e.g., screws, adhesives, etc.).
[0016] The conductive outer shell 64 is designed to receive the pipette tips 66. The conductive outer shell includes sidewalls 80 depending from the sides of a face 82, and a number of tip seats 84 formed upon the face 82. The tip seats are holes in the face 82 and are arranged in a rectangular fashion to form a matrix of tip seats. In the preferred embodiment of the invention, the face 82 and the sidewalls are integrally formed as a single piece to make up the conductive outer shell 64.
[0017] The conductive outer shell 64 is formed of any number of different plastics capable of conducting electricity or dissipating electricity. In one embodiment of the invention, the conductive outer shell is formed mostly of polypropylene and is filled with other material to make the outer shell conductive. For example, the polypropylene may be impregnated with about 15% or more carbon, by weight, to make the plastic conductive. Polypropylene impregnated with about 21% carbon, by weight, is one preferred conductive plastic, such as that manufactured by the RTP Company of Winona, Minn. However, any number of different materials, may be used for the conductive outer shell. For example, in addition to the use of carbon, the plastic may also be made conductive by including metal strands, metal flakes, metal powder in the plastic. Furthermore, instead of impregnating materials into the plastic before the rack is molded to its desired shape, the conductive plastic rack may be formed by coating on the exterior of the rack with conductive plastic material or metallic material.
[0018] The conductive plastic rack is capable of sufficiently conducting or dissipating electricity such that the electricity deposited on a tip placed within the tip rack will flow out of the tip and through the tip rack, thereby prevent the tip from repelling other tips within the rack with any forces significant to disturb the seated position of the other tips within the tip rack. To this end, the tip rack may be grounded to allow static electricity flowing through the tip rack to be grounded. However, if the tip rack is not grounded, the surface area of the rack is sufficiently large to dissipate significant concentrations of static electricity throughout the rack, and avoid any problems wherein pipette tips are dislodged from their seats. Accordingly, the term "conductive plastic" as used herein is intended to cover both electrically conductive plastics and static dissipative plastics.
[0019] The support insert 62 serves as the skeleton for the plastic rack, providing structural support to make the rack 60 strong and sturdy. The support insert 62 is preferably made of polypropylene and may or may not include other material such as graphite to make the insert conductive. Although the support insert 62 is completely covered by the conductive outer shell 64, FIG. 2 shows a cutaway portion of the conductive outer shell that reveals the support insert 62. The insert 62 provides a number of support walls 70 that form chambers 72 for receiving pipette tips 66 when they are inserted into the rack. The support walls 70 provide a lightweight honeycomb structure that allows the face of the conductive outer shell to support a large amount of force. It is important that the face supports a large amount of force because of the substantial force exerted upon the face when the pipettes are attached to the pipette tips sitting in the rack. In addition, the support insert 62 includes a number of buttresses 68 around the outer support walls. These buttresses 68 fit against the sidewalls 80 of the conductive outer shell 64 when the conductive outer shell is joined to the insert 62, thereby providing additional structural support for the conductive outer shell. The two-part rack comprised of the conductive outer shell 64 and the support insert 62 may be manufactured for less cost than a one-piece rack because the more expensive conductive plastic material is only required to form the conductive outer shell 64, and the support insert 62 may be made of less expensive non-conductive plastic.
[0020] Each pipette tip 66 is made of polypropylene and includes a collar portion 90 and a conical body 92. The collar portion 90 includes and interior diameter, and this interior diameter of the collar portion is designed to securely fit around the end of a pipette held by the multiple pipette device and thereby attache the pipette tip to the pipette. The conical body 92 is the portion of the tip that receives and dispenses liquid to be transferred from one location to another. FIG. 2 shows a first portion 94 of the pipette tips removed from their seats 84 and a second portion 96 of the pipette tips positioned within their seats on the tip rack 60. As shown by the first portion 94 of the pipette tips, each pipette tip is placed in the rack by inserting the conical body of the pipette tip into one of the seats 84. As shown by the second portion 96 of pipette tips, when a pipette tip is properly positioned in a seat 84, the collar portion 90 of the pipette tip rests against the face 82 of the conductive outer shell 64 and the conical body 92 is received within one of the chambers 72 of the support insert. Thus, the diameter of each pipette seat 84 is large enough to pass the conical body 92 of a pipette tip, but small enough to prevent passage of the collar portion 90.
[0021] In operation, a robot transfers the rack 60 with seated polypropylene tips 66 to a loading area where a multiple pipette device will pick up the tips positioned within the rack. The head of the multiple pipette device comes into position over the rack 60 and the pipettes are inserted into the collars 90 of the pipette tips. The rubbing of the pipette tips with the collars creates a static electric charge on the tips. After the pipettes and their associated tips have been used to transfer liquids from one location to another, the tips are removed from the pipettes by the shuck plate (not shown). Once again, when the tips are removed from the pipettes, the tip collars 90 rub against the pipettes and electrostatic charges are generated and deposited upon the tips. However, when the tips fall into the seats of the tip rack and come into contact with the conductive outer shell of the rack, the electrostatic charge is conducted from the tips and through the rack to ground or dissipated within the rack. Thus, when the tips are placed in the rack, the tips do not repel against each other or the rack and are held securely in place within the rack by gravity. In this manner, the conductive plastic rack for pipette tips prevents random failures resulting from electrostatic charges on the tips, and therefore makes operation of the multiple pipetting device more reliable.
[0022] Various tests were performed using the conductive plastic tip rack in a laboratory setting. In one test, the conductive outer shell was formed of polypropylene material impregnated with carbon such that the carbon comprised about 21% of the total weight of the polypropylene material. With this 21% carbon, by weight, polypropylene material, the tip rack reliably removed the problem of tips repelling within the tip rack at 17% relative humidity. It was found that in very low humidity testing environments of less than 10% relative humidity, additional humidity is sometimes required to prevent any significant repelling of the tips within the rack using the conductive outer shell formed of polypropylene material having about 21% carbon, by weight. However, other conductive or dissipative outer shells formed of other plastics may perform even more reliably in low humidity conditions.
[0023] Although the present invention has been described in considerable detail with reference to certain preferred versions thereof, other versions are possible. For example, the rack may be a single molded conductive piece and need not be comprised of an insert with a conductive outer shell. As another example, the tips themselves may also be manufactured of a conductive plastic material. Of course, many other versions of the invention are possible. Therefore, the spirit and scope of the invention should not be limited to the description of the preferred versions contained herein.
|Cited Patent
|Filing date
|Publication date
|4 May 1936
|28 Mar 1939
|American Box Board Co
|Title not available
|Title not available
|Title not available
|Title not available
|Citing Patent
|Filing date
|Publication date
|10 Jan 2002
|30 Jan 2007
|Molecular Bioproducts, Inc.
|Pipette tip reloading system
|4 Sep 2008
|21 Jun 2011
|Jvr Scientific Llc
|Apparatus for handling pipet tips
|17 Jan 2007
|4 Oct 2011
|Argos Therapeutics, Inc.
|Systems and methods for processing samples in a closed container, and related devices
|3 Jul 2012
|Argos Therapeutics, Inc.
|Methods for processing samples in a closed container
|1 Aug 2008
|25 Sep 2012
|Polyone Corporation
|Electrically conductive polyolefin blends
|7 Jun 2012
|30 Apr 2013
|Argos Therapeutics, Inc.
|Apparatus for transfer of liquid for processing samples
|22 Jan 2010
|25 Jun 2013
|Biotix, Inc.
|Anti-static pipette tip trays
|30 May 2012
|5 Aug 2014
|Biotix, Inc.
|Integrated pipette tip devices
|18 Mar 2013
|26 May 2015
|Argos Therapeutics, Inc.
|Apparatus for measuring volume of a fluid
|15 Feb 2013
|28 Jul 2015
|Biotix, Inc.
|Anti-static pipette tip trays
|21 Feb 2013
|11 Aug 2015
|Biotix, Inc.
|Pipette tips
|21 Feb 2013
|18 Aug 2015
|Biotix, Inc.
|Pipette tip rack
|13 Jan 2004
|23 Jun 2005
|Brand Gmbh And Co Kg
|Packing box for laboratory articles
|27 May 2004
|1 Dec 2005
|Gard Douglas J
|Pipetting system with selective pipette tip loading
|2 Sep 2010
|Biotix, Inc.
|Anti-static pipette tip trays
|16 Dec 2010
|27 Oct 2011
|Stanley Preschutti
|Antimicrobial fluid handling devices and methods of manufacture
|17 Jan 2007
|14 Feb 2008
|Argos Therapeutics Inc
|Systems and methods for processing samples in a closed container, and related devices
|20 Feb 2014
|28 Aug 2014
|Biotix, Inc.
|Pipette tip rack
|U.S. Classification
|International Classification
|Cooperative Classification
|European Classification
|8 Feb 2002
Owner name: BECKMAN COULTER, INC., CALIFORNIA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:MORITZ, JEFFREY R.;AVNET, ALLAN;REEL/FRAME:012583/0721
Effective date: 20020201
|22 Nov 2010
|Fee payment
Year of fee payment: 4
|24 Nov 2014
|Fee payment
Year of fee payment: 8